Manufacture of polyoxygenated dehydro-steroids



United States Patent 0 MANUFACTURE OF POLYOXYGENATED DEHYDRO-STEROIDS Albert Wettstein and Ernst Vischer, Basel, and Charles Meystre, Arlesheim, Switzerland, assignors to Ciba Pharmaceutical Products Inc., Summit, NJ.

No Drawing. Filed Feb. 21, 1956, Ser. No. 566,777

Claims priority, application Switzerland Feb. 25, 1955 14 Claims. (Cl. 195-51) This invention relates to the manufacture of polyoxygenated dehydro-steroids by dehydrogenation and introduction of oxygen in various positions by a biochemical method.

It is already known to inroduce oxygen into particular positions in steroids, especially in ll-position, with the aid of micro-organisms (cf. e.g. U.S. Patent No. 2,602,769). In U.S. patent application Serial No. 500,994, filed April 12, 1955, by Albert Wettste'n et al., a process is described according to which it is possible by the use of specific fungi, to oxygenate steroids in 17-position. According to U.S. patent appli ation, Serial No. 518,922, now Patent No. 2,778,776, filed June 29, 1955, by Albert Wettstein et 211., it is further possible by the biochemical method to introduce oxygen into the 2l-position of steroids. It is also known that by means of cultures of specific fungi steroids can be oxygenated simultaneously in different positions, for example in 6- and ll-position. According to U.S. patent appl'cation, Serial No. 563,437, filed February 6, 1956, by Albert Wettstein at al., now Patent No. 2,949,405, by the microbiological method it is further possible to introduce into steroids the 1:2- and if desired the 4:5 double bond without side chain degradation or ring splitting. It has not hitherto been possible, however, in one operation by the biochemical method to introduce oxygen into at least one of the important positions 11, 17 and 21 and a double bond in 1:2- and/or 4:5-position. Such a process would obviously be of great technical importance.

The present invention is based on the observation that polyoxygenated dehydro-steroids can be produced in a simple manner when steroids which are saturated in 1:2- and/or 4:5-position and which are not oxygenated in at least one of the positions 11, 17 and 21, are subjected to the action, in one o-peration, of enzymes from aerobic cultures of Calonectria decora, Ophiobolus heterostrophus, Ophiobolus miyabeanus, Alzernaria passiflorae, or Didymella lycopersici, which are ca able of effecting dehydrogenation in 1:2- and/or 4:5-position and of enzymes from aerobic cultures of at least one of a group of three fungus strains which introduce oxygen in 11-, 17- or 21-position.

The starting materials for the new process are steroids which are saturated in 122- and/or 4:5 position, for example derivatives of spirostane, allosp'rostane, furostane, allo-furostane, cholane, allocholane, androstane or testane, preferably compounds of the pregnane series, among which are to be understood derivatives of any configuration of 10: 13-dimethyl-17-ethyl-cyclopentanopolyhydrophenanthrene and also its higher and lower homologues, for example corresponding A-nor-, D-ho-moand 19-nor-compounds. They can be saturated or contain double bonds, for example in 1- or 4-position or also in 5-, 6-, 7-, 8-, 9-, 11-, 14-, 15- and/or l6-position. The configuration of the starting materials is especially that of pregnane, Set-pregnane, 17a-pregnane or corresponding racemates such as are obtained in total synthesis. As such starting materials there are used pri- 2,969,305 Patented Jan. 24, 1961 marily compounds which are saturated in 1:2- and/or in 4:5-position and oxygenated in 3- and ZO-position and if desired in l8-position and/or in one of the positions 11, 17 and 21 and their functional derivatives, that is to say pregnanes which contain in the specified positions a free or functionally converted oxo or hydroxyl group, as for example esters, ethers, thioethers, thiol or thione esters, acetals, mercaptals, keta's, enol d.r vatives, such as enol esters or enamines, hydrazones, semicarbazones and the like. They can also be further substituted, as for example by free or functionally converted hydroxyl, 0x0 or epoxy groups, for example in 6-, 7-, 9-, 11-, 12-, 15-, 16- or 19-position, and especially by halogen atoms, such as chlorine or fluorine in 9-position or by a methyl group in l7u- 0r 17,8-position. Specific starting materials include progesterone, Hot-progesterone, 16a-hydroxy-, 17a-hydroxy-, 18-hydroxyor 18 0x0- progesterone, cortexone, 18-hydroxyor l8-oxo-cortexone, ll-keto-progesterone, llaandllfl-hydroxy progesterone, 9:11- or 11:12-dehydro-progesterone, 19-hydroxy-progesterone, 9-chloroor 9-flucro-11fl-hydroxyprogesterone, 115218 dihydroxy progesterone, llflhydroxy-l8-oxo-progesterone, 9-chloroor 9-fiuor0-llfihydroxy-l8-oxo-progesterone, 11:18 dioxo progesterone, 19-nor-progesterone, 19-nor-11p hydroxy-18-oxo-progesterone, corresponding compounds unsaturated in l-position instead of in 4-position, pregnenolone, A -pregnene- 3 20-diols, pregnane-3 :ZO-dione, pregnene-B-ol-ZO-one, allopregnane-3z20-dione, pregnane-3z11z20-trione, allopregnane-3:1l:20-trione-17u-ol, or their functional derivatives.

The enzymes to be used for the oxidation in ll-position are preferably produced from aerobic cultures of fungi o-f the genera Rhizopus, Mucor, Aspergillus, Penicillium, Curvularia, Cunninghamella, Spondylocladium or Streptomyces. For the oxidation in 17-positio-n there are suitable enzymes from aerobic cultures of T richo thecium roseum, Leptosphaeria maculans, Cucurbitaria laburni, Acrospeira levis, Lophotrichus martinii, Melaospam parasitica o-r Thielavia terricola. The enzymes for the oxidation in 21-position are advantageously obtained with the aid of aerobic cultures of Ophiobolus herpotrichus or Sclerotinia fructicola. The dehydrogenation in 1:2- and/or in 4:5-position is carried out with enzymes of Didymella lycopersici, Calonectria decora, Alternaria passz'florae, Ophiobolus heteroszrophus, or Ophiobolus miyabeanus. In the process it is possible to work with isolated or concentrated enzyme preparations, but especially with crude growing fungus cultures, with their filtrates or with mycelium suspen- The culture solutions used for the culture of these fungi are suitably agitated, i.e. shaken or stirred and contain assimilable carbon, especially carbohydrates, such as glucose or sucrose and also if desired growth promoting materials, for example corn steep liquor or beer wort and inorganic salts. Thus natural, synthetic or semi-synthetic nutrient solutions can be used. Such nutrient solutions are advantageously employed as offer to all the fungi used optimum growth promoting conditions. It is also possible to add later specially suitable nutrient and growth promoting substances for the fungi, for example yeast extract.

The process is carried out in one operation. It has however proved to be advantageous to carry out the dehydrogenation and oxygenation at the specified posi ions consecutively. The simplest practical process is described below, but other processes can also be used within the scope of the invention.

The organism required for the first oxygenation or the dehydrogenation iscultivated in similar apparatus and under similar conditions to those known in antibiotic manufacture as the so called deep tank process for instance in that the sterilized nutrient solution eg. beer wort, is inoculated with the microorganism, and then shaken or stirred and aerated for some days, prefrably at a temperature of about 27 C. After develcpfnent of the cultures, the specified starting materi ls, such as e.g. progesterone or cortexone are introduced in fine dispersion or solution, for example in met anol, acetone or ethylene glycol and incubation is contin ed until the maximum reaction is attained. Then, wihout previous filtration or isolation of the reaction pr duct, there is added to the react'on mixture a developed culture of the second organism, and if necessary cor esponding nutrient and growth promoting substanc s and the incubation is then continued, generally for a f w days longer. If desired, this cperation is repeated with a further microorganism. The course of the dehydro 'genation and of the individual oxygenations can be followed by paper chromatography. Subsequenly the liquor is separated from the medium and the filtrate and/or the mycelium mass extracted and from the ex tract the reaction products isolated in the conven it) al manner, for example by separation processes, adsorption, chromatography, crystallization, conversion into farctional derivatives, such as Glrard compounds and the like.

The fungus cutures or enzymes can be added in any convenient sequence. By preliminary experiments it can easily be ascertained which sequence offers advantages in any particular case.

According to the present process valuable medicaments are obtained of the seroid and especial'y of the pegnane series, which are distinguishable frcm the teapeutically active compounds which are saturated in [:2- position by an increased activity. Of the products of the process there may be named especially A 3:11:20 trioxo l7a:2l dihydroxy pregnadiene, A 3 dioxo 11B: 170::21 trihydroxy pregnadiene, A 3: ll: 20 trioxo 21 hydroxy pregnad'ene, A 3:20- dioxo 115:21 dihydroxy pregnadiene, A 3:20- dioxo 171x121 dihydroxy pregnadiene, A 3:"0- dioxo 21 hydroxy prcgnadiene, A 3:18:20 trioxo llflz21 dihydroxy pregnadiene, A 3:11:18: 20 tetraoxo 21 hydroxy pregn diene, A 3:20- dioxo 11 8: 18:21 trihydroxy pregnadiene, A 3 18: 20 trioxo 115: 170::21 trihydroxy pregnadiene, A 3:1l:18:20 tetraoxo 1701221 dihydroxy pregnadiene, A 3:20 dioxo 11 8: 170:: 18:21 tetrahydroxy pregnadiene, A 3:20 dioxo 18:21 dihydroxy pregnadiene, A 3: 18:20 trioxo 21 hydroxy pregnadiene, A 3:20 dioxo 170::18221 trihydrixypregnadiene, A 3: 18:20 trioxo 170;:21 dihydr xypregnadiene, the corresponding 21-oxoand 21-desoxycompounds, and also correspond'ng functional d'rivatives, such as esters, ethers, halogen derivatives, for example 9a-halogen compounds, especially the fluorine or chlorine compounds. Insofar as the prcducts of the process do not possess the configuration or the substituents of therapeutically useful steroids, they can be used as intermediate products for the manufacture of such compounds, for exampe of compounds as specified above.

The reaction products resuting from the process of this invention can be converted in the conventional manner into their functional derivatives, such as oxygen, 'ulphur or nitrogen derivatives, for example esters, ethers, enol esters, enol ethers, ketals, thioe'hers, and thioketals, or also hydrazones, oximes or enamines. In these compounds, the hydroxyl and/or oxo groups can be completely or partially functionally converfed.

In the esters and cool esters the acid residues can be those of any organic or inorganic acds, such as aliphatic, alicyclic, araliphatic, aromatic or heterocyclic cal-boxy ic, thione carboxylic, thiol carboxylic or sulphonic acids, preferab'y of formic acid, acet'c acid, chloracetic acids,

trifluoroacetic acid, propionic acid, butyric acids, valeric acids, trimethylacetic acid, diethylacetic acid, caproic acids, oenanthic acids, caprylic acids, palmitic acids, crotonic acid, undecanic acid, undecylenic acid, oxalic acid, succinic acid, pimelic acid, maleic acid, lactic acid, carbamic acids, alkoxy-carboxylic acids, fl-cyclopentyl-propionic acid, hexahydroberizoic' acid, benzoic acid, phenylacetic acid, cyclohcxyl acetic acid, phenylpropionic ac ds, trimethy -ga1lic acid, phthalic acid, furane-Z-carbdxylic acid, isonicotinic acid, methane sulphonic acid, toluene sulphonic acid, sulphuric acids, hydrohalic acids or phosphoric acids.

If desired, in resulting comptunds, functionally ccnverted hydroxyl or oxo groups can be converted i.'.to free groups. In this manner, especially in polysubsitu'ed derivatives, the functionally converted groups can zlso be partially liberated. This can take p ace, for example, by chemical or enzymatic hydrolysis, for example us ng acid or basic agents, by re-esterification or re-acetalzation. From the only partially converted, for example esterified or etherified derivatives, obtained in this manner or also direcily, there can be produced by subsequent functional conversion, for example esterification or etherification, polysubstituted derivatives, especially mixed esters or ethers or ester-ether.

9:11;3-ox do-compounds which are obtained can be converted by reaction with hydroha ic ac'ds, especial'y hydrofluoric and hydrochloric acid, into the corresponding 9: 1 l-halogenhydrins.

The final products of this application such as l-dehy dro-hydrocortisone, l-dehydro-cortisone, 1-dehydroco;ticosterone, l-dehydro-ll-desoxv-corticosterone etc. possess known therapeutic utilities. They are of value in the treatment of various diseases in humans and lower animals involving metabolica'nd glandular disturbances. Their doses, methods of administration and particular physiological properties are known and described in the literature.

The following examples illustrate the invention.

Example 1 In a shaking vessel of 16 liters capacity, 3.6 liters of beer wort are sterilized and inoculated with 400 cc. of a two day old shaking culture of Ophiobolus herpotrichus grown in beer wort. The whole is shaken for two days at 27 C., whereby the culture becomes well developed. Then a solution of 1.0 gram of progesterone in 25 cc. of acetone is added under sterile conditions. At the same time, in a second shaking vessel, 3.6 liters of sterile beer wort are inoculated with 400 cc. of a two day old shaking culture of Calonectria decor-a, likewise grown in beer wort. The two vessels are shaken for three days at 27" C. The Callonectria-culture which is now fully developed, is transferred under sterile conditions to the 16 liter vessel, which is now shaken for a further two days at 27 C. The mycelium is then separated and the culture filtrate extracted by shaking four times, each time with 2 liters of ethyl acetate. The extracts are washed three times, in each case with 300 cc. of water, dried and evaporated under vaccum. The resulting residue (1.4 grams) is dissolved in 160 cc. of methanol, 40 cc. of water added and the whole extracted by shaking three times with 50 cc. of pentane each time. The pentane extracts only contain oily impurities, the steroids remaining in the methanolic solution. This is evaporated under vacuum at 30 C. and dried under high vacuum. The residue is chromatographed on 30 grams of silica gel by the fractional elution method, elution being carried out first with chloroform, then with chloroform-acetone mixtures of increasing acetone content and finally with ace tone. The individual fractions (each of 100 cc.) are examined by paper-chromatography. The fractions eluted with chloroform contain together with impurities some starting material and the chloroform-acetone (97.5 :2.5 mixtures some cortexone (ll-desoxy-corticosterone). The majority of the substance is contained in the chloroform-acetone (:5) fractions and consists for smarts the most part of l-dehydrocortexone, which is obtained in crystalline form from an acetone-petroleum ether mixture; M.P. 185193 C.; [a] =+120 (CI-ICI Ultra-violet absorption spectrum (in ethanol) A max, 244 m, (e=14,100). Example 2 In an Erlenmeyer flask of 500 cc. capacity, 50 cc. of beer wort are sterilized and inoculated with Ophiobolus herpotrichus. The culture is shaken at 27 C. After three days it is well developed and there is added thereto under sterile conditions a solution of 15 mg. of progesterone in 0.75 cc. of acetone. At the same time in a second Erlenmeyer flask 50 cc. of beer wort are sterilized and inoculated with a strain of Curvularia brachyspora. The two cultures are now shaken in the same way at 27 C. After three days the completely developed Curvularia culture is added under sterile conditions to the Ophiobolus culture. The combined cultures are further shaken. At the same time in a third Erlenmeyer flask 50 cc. of sterile beer wort are inoculated with Calonectria decora and shaken at 27 C. After three days this Calonectria cultureis added under sterile conditions to the mixture of the two other cultures, whereupon the whole is further shaken at 27 C. After three days, the mycelium is separated off and the culture filtrate extracted by shaking three times with 30 cc. of ethyl acetate each time. The extracts are washed with water, dried and evaporated. Paper chromatographic examination of the residue shows the presence of l-dehydro-l15:2l-dihydroxyprogesterone (l-dehydro-corticosterone If in the above process the progesterone is replaced by 18-oxo-progesterone, l-dehydro-aldosterone is obtained.

Example 3 'As described in Example 2, to a culture of Ophiobolus herpotrichus in 50 cc. of beer wort a solution of 15 mg. of progesterone in 0.75 cc. of acetone is added. At the same time, in a second Erlenmeyer flask 50 cc. of sterilized beer wort are inoculated with Trichothecium roseum. The two cultures are now shaken at 27 C. After three days, with avoidance of infection, they are combined and the mixture is shaken for a further three days at the same temperature. At the same time in a third Erlenmeyer flask 50 cc. of sterile beer wort are inoculated with Calonectria decora and shaken at 27 C. After three days this Calonectria culture is added under sterile conditions to the mixture of the two other cultures, whereupon the whole is further shaken at 27 C. After three days, the mycelium is separated off and the culture filtrate extracted as described in Example 1. The extraction residue is shown by paper chromatographic examination to contain 1-dehydro-17a:2l-dihydroxy-progesterone.

Example 4 By replacing in Example 3 the progesterone by ll-keto progesterone and incubating in the manner described with cultures of Ophiobolus herpotrichus, Trichothecium roseum and Calonectria decora, a considerable quantity of 1 dehydro-ll-keto-17a:2l-dihydroxy-progesterone (1- dehydro-cortisone) can be detected in the extraction residue.

Example 5 By replacing in Example 3 the progesterone by 11,3- hydroxy-progesterone and incubating in the same way with cultures of Ophiobolus herpotrichus, Trichothecium roseum and Calonectria decora, considerable quantities of l-dehydro-l 1B:17a:2l-trihydroxy-progesterone (l-dehydro-hydrocortisone) can be detected in the extraction residue.

Example 6 As described in Example 2, there is added under sterile conditions to a culture of Ophiobolus herpotrichus in 50 cc. of beer wort, a solution of 15 mg. of progesterone in"0.75 cc. of acetone. At the same'time, in a second Erlenmeyer flask 50 cc. of sterile beer wort are inoculated with Leptosphaeria maculans. The two cultures are shaken at 27 C. and after three days are combined. At the same time, in a third Erlenmeyer flask 50 cc. of sterile beer wort are inoculated with Curvularia lanata. This culture and the previously mentioned culture mixture are shaken for three days at 27 C. as a result of which the Curvularia culture becomes well developed. It is then united under sterile conditions with the culture mixture. The whole is shaken for a further three days at 27 C. At the same time in a fourth Erlenmeyer flask 50 cc. of sterile beer wort are inoculated with Alternaria passiflorae and shaken at 27 C. After three days this Alternaria culture is added under sterile conditions to the mixture of the three other cultures, whereupon further shaking is conducted at 27 C. The mycelium is then separated oil and the culture filtrate extracted as described in Example 1. The paper chromatographic examination of the extraction residue shows the presence of considerable quantities of l-dehydro-llfl:17a:ZI-trihydroxy-progesterone (1-dehydro-hydrocortisone) together with l-dehydro- 11 keto-17a:21-dihydroxy-progesterone (l-dehydro-cortisone).

Example 7 When the sequence of the additions of the specified cultures as described in Example 6 is varied in such a manner that the progesterone is first incubated with a culture of Curvularia lunata, then in the described manner a culture of Ophiobolus herpotrichus added, then a culture of Alternaria passiflorae and finally one of Leptosphaeria maculans, the extraction residue obtained in the customary manner likewise contains l-dehydro-l 1,3: 17a:21-trihydroxy-progesterone l-dehydro-hydrocortisone) and l-dehydro-l1-keto-l7az2l-dihydroxy-progesterone l-dehydro-cortisone) Example 8 50 cc. of sterile beer wort are inoculated with Cunninghamella blakesleeana. The culture is shaken for two days at 27 C. and then, under sterile conditions, a solution of 15 mg. of ll-desoxy-corticosterone (cortexone) in 0.75 cc. of acetone is added. At the same time, in a second Erlenmeyer flask 50 cc. of sterile beer wort are inoculated with Trichothecium roseum.

The two cultures are shaken separately for a further two days at the same temperature and then combined under sterile conditions and shaking continued. At the same time, in a third Erlenmeyer flask 50 cc. of sterile beer wort are inoculated with Calonectria decora and shaken at 27 C. After three days this Calonectria culture is added under sterile conditions to the mixture of the two other cultures, whereupon shaking is further continued at 27 C. After two days the mycelium is separated off. The extraction of the culture filtrate takes place in the same manner as described in Example 2. The extraction residue contains l-dehydro-l 1fi3:17a:2ltrihydroxy-progesterone (1-dehydro-hydrocortisone) together with l-dehydro-ll-keto-l7a:21-dihydroxy-progesterone l-dehydro-cortisone).

Example 9 In a shaking vessel of 18 liters capacity 3.6 liters of beer wort are sterilized and inoculated with 400 cc. of a two days old shaking culture of Ophiobolus herpotriclzus which has been grown in beer wort. Shaking is carried on for two days at 27 C., whereby the culture becomes well developed. Then a solution of 1.0 gram of progesterone in 25 cc. of acetone is added under sterile conditions. At the same time, in a second shaking vessel 3.6 liters of sterile beer wort are inoculated with 400 cc. of a three days old shaking culture of Trichothecium roseum, likewise cultivated in beer wort. The two vessels are shaken for three days at 27 C. The Trichothecium culture, which is now fully grown, is transferred under sterile conditions to the 18 liter vessel 7 and at the same time, in a further shaking vessel. 3.6 liters of sterile beer wort are inoculated with 400 cc. of a 24 hours old shaking culture of Calonectria decora grown in beer wort. This shaking vessel, and also that which contains the culture mixture just described above, are shaken for three days at 27 C. and their contents are combined under sterile conditions. At the same time 3 liters of sterile beer wort are inoculated with 400 cc. of a 36 hours old shaking culture of Curvularia lunata grown in beer wort. This culture, and also the culture mixture in the 18 liter flask, are shaken for two days at 27 C. whereupon their contents are combined under sterile conditions. Shaking is continued for two days longer at the specified temperature and then the mycelium is separated olf. The culture filtrate is extracted by shaking four times, in each case with three liters of ethyl acetate. The extracts are washed three times, with 500 cc. of water in each case, dried and evaporated. The residue obtained (1.1 grams) is dissolved in 160 cc. of methanol, 40 cc. of Water added and the whole extracted by shaking three times, using 50 cc. of pentane each time. The pentane extracts only contain oily impurities, while the steroids remain in the methanolic solution. This is evaporated under vacuum at 30 C. and dried under high vacuum. The residue is chromato-graphed on 30 grams of silica gel by the fractional elution method, elution being carried out first with methylene chloride, then with chloroform and with chloroform-acetone mixtures and finally with acetone. The individual fractions (150 cc.) are examined by paper chromatography. The fractions eluted with methylene chloride and with chloroform contain impurities, some starting material and l1-desoxy-corticosterone, while in the mixtures of chloroform and acetone (9:1 and 8:2) l-dehydro-ll-keto-l7oz:21-dihydroxy-progesterone (1-dehydro-cortisone) is contained. The fractions concerned are evaporated and the l-dehydro-cortisone is obtained after recrystallization from a mixture of acetone and isopropyl ether, in crystals of M.P. 230- 233 C. The chloroform-acetone (1:1) fractions contain 1-dehydro-1 113: 17a:21-trihydroxy-progesterone (l-dehydro-hydrocortisone), which crystallizes from acetonepetroleum ether; M.P. 238-240 C.

Example 10 By using in the method of Example 9 instead of the culture of Calanectria decora a culture of Didymella lycopersici grown in a similar manner and proceeding otherwise as described in Example 9, there are likewise obtained l-dehydro-l1-keto-17az2l-dihydroxy-progesterone (l-dehydro-cortisone), M.P. 230-233 C. and l-dehydro-11B: 171x:21-trihydroxy-progesterone (l-dehydrohydrocortisone), M.P. 238-240" C.

Example I] Quantities of 3.6 liters each of terilized beer wort are separately inoculated with Ophiobolus herpotrichus, Trichothecium rosezzm, Didymella Iycopersici and Curvularia lurmta and shaken at 27 C. After three days the cultures are combined and there is added to the mixture a solution of 1.0 gram of progesterone in cc. of acetone whereupon the Whole is further shaken at 27 C. After four days the mycelium is separated. As described in Example 9, the culture filtrate is extracted and the extraction residue chromatographed on silica gel. Again l-dehydro-cortisone of M.P. 230-233 C. and l-dehydro-hydrocortisone of M.P. 238-240 C. are obtained.

Example 12 Quantities of 3.6 liters each of sterilized beer wort are separately inoculated with Ophiobolus herpotrichus, Tricholhecium roseum, Didymella lycopersici and C urvularia lunata and shaken at 27 -C. After three days the four cultures are combined and the mycelium is separated til ed and suspended. in 5 liters of water. A solution of 1.0 gram of progesterone in 25 cc. of acetone is added and the whole is shaken at 27 C. After four days the suspension is filtered ofi. As described in Example 9, the filtrate is extracted and the extraction residue chromatographed. There are obtained l-dehydro-cortisone of M.P. 230-233 C. and l-dehydro-hydrocortisone of M.P. 238-240 C.

Example 13 cc. of sterilized beer wort are inoculated with T richothecium roseum and shaken for three days at 27 C. Then a solution of 40 mg. of 9:11 3-oxido-cortexone- 21-acetate in 1.5 cc. of acetone is added. At the same time 120 cc. of sterile beer wort are inoculated with Dia'ymella Iycopersici. Both cultures are shaken at 27 C. After three days they are combined and shaken for a further three days at 27 C. Then the mycelium is filtered off and the culture filtrate extracted by shaking three times with 50 cc. of ethyl acetate each time. The combined extracts are washed with water, dried and evaporated under vacuum. The extraction residue (42 mg), according to paper chromatographic examination, consists for the most part of l-dehydro-9:llfi-oxido-lhhydroxy-cortexone. This is purified by preparative paper chromatography (system propylene glycol-toluene) and acetylated with 4 cc. of a mixture of pyridine and acetic anhydride in the customary manner. The resulting crude product is dissolved in 5 cc. of dioxane, mixed with 1.25 cc. of 2.5 N hydrofluoric acid in chloroform and the whole allowed to stand for one hour at room temperature. Water is then added and the whole extracted with chloroform-ether (1:3). After washing with water, drying and evaporation of the solvent under vacuum l-dehydro-9afluoro-hydrocortisone-Zl-acetate is obtained. It is recrystallized from a mixture of acetone and petroleum ether; M.P. 235-277 C.

Example 14 120 cc. of steriized beer wort are inoculated with Trichothecium roseum and shaken for three days at 27 C. Then a solution of 40 mg. of 9a-fluoro-corticosterone- 21-acetate in 1.5 cc. of acetone is added. At the same time 120 cc. of sterile beer wort are inoculated with Didymella lycopersici. The two cultures are separately shaken at 27 C. After three days they are combined and shaking continued at 27 C. for a further three days. The mycelium is then separated off and the culture filtrate extracted by shaking three times, in each case with 50 cc. of ethyl acetate. The combined extracts are washed with water, dried and evaporated under vacuum. The extraction residue (42 mg), according to paper chromatographic examination, consists for the most part of 1-dehydro-9a-fiuoro-hydrocortisone. This is purified by means of a preparative paper chromatogram using the system propylene glycol-toluene. The resulting crude product is dried under high vacuum at 40 0., dissolved in 2 cc. of pyridine and treated with 2 cc. of acetic anhydride. After 16 hours standing the Whole is poured onto ice. The precipitated crystalline product is filtered otf, washed with water, dried and recrystallized from a mixture of acetone and petroleum ether. The resulting 1-dehydro-9a-fluoro-hydrocortisone-21-acetate melts at 235-237 C.

What is claimed is:

1. In a method for dehydrogenating the 1:2-position and oxygenating at least one of the positions 11, 17 and 21 in a A' -3:20-dioxo-pregnene, saturated in the 1:2-position and nonoxygenated in at least one of the positions 11, 17 and 21, the step which comprises subjecting a A -3:20-dioxo-pregnene, saturated in the 1:2-position and non-oxygenated in at least one of the positions 11, 17 and 21, simultaneously to enzymes from aerobic cultures of fungi selected from the group consisting of Caionectria decora, Ophiobolus heterostrophus, Ophiobalus miyabeanus, .Altemaria passiflorae and .Didymella Jycqpersici for dehydrogenation of the 1:2-position, and to at least one type of enzymes produced from one of the three groups of fungi consisting of (a) the genera Rhizopus, Mucor, Aspergillus, Penicillium, Curvularia, Cunninghamella, Spondylocladium and Streptomyces for oxidation in position 11, (b) the species Trichothecium roseum, Leptosphaeria maculans, Cucurbitaria laburni, Acrospeira levis, Lophotrichus martinii, Melanospora parasitica and Thielavia Ierricola for oxidation in position 17 and (c) the species Ophiobolus herpotrichus and Sclerotinia fructicola for oxidation in position 21, said treatment being carried out in a single operation.

2. A process as set forth in claim 1, wherein the enzymes used are in the form of the crude submerged culture.

3. A process as set forth in claim 1, wherein the enzymes used are in the form of the filtrate of the submerged culture.

4. A process as set forth in claim 1, wherein the enzymes used are in a substantially concentrated form.

5. A process as set forth in claim 1, wherein the starting material is progesterone.

6. A process as set forth in claim 1, wherein the starting material is ll-keto-progesterone.

7. A process as set forth in claim 1, wherein the starting material is 1lfi-hydroxy-progesterone.

8. A process as set forth in claim 1, wherein the starting material is cortexone.

9. A process as set forth in claim 1, wherein the starting material is 18-oxo-progesterone.

10. A process as set forth in claim 1, wherein the starting material is pregnane-3z11z20-trione.

11. A process as set forth in claim 1, wherein the starting material is allopregnane-3:llz20-trione-l7a-ol.

12. A process as set forth in claim 1, wherein the starting material is 9u-fluor-corticosterone.

13. A process as set forth in claim 1, wherein the starting material is 9:1lB-oxido-cortexone acetate.

14. A process as set forth in claim 1, wherein the starting material is a 9:11fl-oxido-pregnane compound and the resulting 9:11fi-oxido compound is treated with a hydrohalic acid to obtain the corresponding 9:11-halogenhydrin.

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Meister, P., et al.: Jour. Am. Chem. Soc. 76, August 5, 1954, pages 4050, 4051.

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1. IN A METHOD FOR DEHYDROGENATING THE 1:2-POSITION AND OXYGENATING AT LEAST ONE OF THE POSITIONS 11, 17 AND 21 IN $4-3:20-DIOXO-PREGNENE, SATURATED IN THE 1:2-POSITIONS TION AND NONOXYGENATED IN AT LEAST ONE OF THE POSITIONS 11, 17 AND 21, THE STEP WHICH COMPRISES SUBJECTING A $4-3:20-DIOXO-PREGNENE, SATURATED IN THE 1:2-POSITION AND NON-OXYGENATED IN AT LEAST ONE OF THE POSITIONS 11, 17 AND 21, SIMULTANEOUSLY TO ENZYMES FROM AEROBIC CULTURES OF FUNGI SELECTED FROM THE GROUP CONSISTING OF CALONECTRIA DECORA, OPHIOBOLUS HETEROSTROPHUS, OPHIOBOLUS MIYABEANUS, ALTERNARIA PASSIFLORAE AND DIDYMELLA LYCOPERSICI FOR DEHYDROGENATION OF THE 1:2-POSITION, AND TO AT LEAST ONE TYPE OF ENZYMES PRODUCED FROM ONE OF THE THREE GROUPS OF FUNGI CONSISTING OF (A) THE GENERA RHIZOPUS, MUCOR, ASPERGILUS, PENICILLIUM, CURVULARIA, CUNNINGHAMELLA, SPONDYLOCLADIUM AND STREPTOMYCES FOR OXIDATION IN POSITION 11, (B) THE SPECIES TRICHOTHECIUM ROSEUM, LEPTOSPHAERIA MACULANS, CUCURBITARIA LABURNI, ACROSPEIRA LEVIS, LOPHOTRICHUS MARTINII, MELANOSPORA PARASITICA AND THIELAVIA TERRICOLA FOR OXIDATION IN POSITION 17 AND (C) THE SPECIES OPHIOBOLUS HERPORTICHUS AND SCLEROTINIA FRUCTICOLA FOR OXIDATION IN POSITION 21, SAID TREATMENT BEING CARRIED OUT IN A SINGLE OPERATION. 